The immune system provides the major host defense against intracellular pathogens and tumors. CD8 T lymphocytes acquire the ability to specifically discriminate infected and malignant cells from normal cells during positive selection in the thymus. CD8 T cells emerge with the ability to specifically and vigorously react with self MHC to which a foreign antigenic peptide is bound, yet remain tolerant to self MHC bound by self peptides. The majority of studies agree that positive selection involves combinatorial recognition by TCR of a peptide/MHC complex yet the nature of the specific peptide/MHC ligand that drives positive selection remains elusive and controversial. The role of peptide in positive selection of the T cell repertoire is at the heart of several unresolved issues. If peptide is involved during development, how does it impact on the peptide specificity of peripheral CD8 T cell activation? Furthermore, the nature of the peptides involved and their relationship to the antigenic peptides remains a matter of controversy. In addition, the extent to which a single peptide/MHC can drive positive selection is still highly controversial. For example, an issue that remains hotly debated is whether specific interaction with any one self peptide selects a limited or diverse set of TCRs. Although several elegant previous reports have addressed these questions, in particular using the OVAp/Kb and VSVp/Kb systems, it has been difficult to extend in vitro findings using organ culture to in vivo models because of the enormity of the pool of self peptides. Thus, defining the relationship between the peptide/MHC complexes that drive positive selection and the selected repertoire remains a challenge. However we have recently developed unique mouse strains that express only the OVAp/Kb complex or the VSVp/Kb complex. Using rigorous clonal T cell approaches coupled with isolation of Kb self peptides and extensive tests of peptide specificity, experiments proposed in this application will define the role of peptide in CD8 T cell selection. These studies will address unresolved questions regarding CD8 T cell development. We will determine how selection on a single peptide/MHCI complex influences the size and diversity of the CD8 T cell repertoire. This analysis will allow us to determine if positive selection is indeed peptide specific. We will establish the relationship between the selecting and cognate peptides and we will determine the impact of structural similarity on the development and function of the CD8 T cell repertoire.